Inactivated enzyme variants and associated process and reagent system

ABSTRACT

The present invention relates to oxidoreductase apoenzyme variants which are enzymatically inactive but have coenzyme-binding properties. Further, the present invention relates to DNA sequences encoding these oxidoreductase apoenzyme variants, expression vectors containing such DNA sequences and the use of these oxidoreductase apoenzyme variants in diagnostic applications.

TECHNICAL DESCRIPTION

The present disclosure relates to oxidoreductase apoenzyme variantswhich are enzymatically inactive but have coenzyme-binding properties.Further, the present invention relates to DNA sequences encoding theseoxidoreductase apoenzyme variants, expression vectors containing suchDNA sequences and the use of these oxidoreductase apoenzyme variants indiagnostic applications.

BACKGROUND

It is known in the art to use enzymatic methods to detect analytes, forexample glucose in blood. In these methods, the analyte to be detectedis contacted with a detection reagent which contains a coenzyme. Thecoenzyme is reduced or oxidized upon enzymatic oxidation or reduction ofthe analyte, respectively. For highly concentrated analytes this changein the redox state of the coenzyme can be measured directly, e.g. bydual UV-wavelength measurement. However, if the concentration of theanalyte and thus the concentration of the coenzyme is below about 10⁻³M, enzymatic detection methods frequently require that the redoxequivalents formed upon oxidation or reduction, respectively have to betransferred to mediators which are then detected electrochemically orphotometrically in a further step. Enzymatic detection methods thatutilize mediators require a calibration step which furnishes a directconnection between the measured value and the concentration of theanalyte to be measured. In light of the above discussion, additionaloptions for detecting analytes in a sample are desirable.

SUMMARY OF THE INVENTION

Embodiments of the invention in accordance with the present disclosuremay comprise one or more of the following features or combinationsthereof;

The disclosure provides a variant of an oxidoreductase apoenzyme whichis (i) substantially enzymatically inactive and (ii) capable ofefficiently binding a coenzyme.

The disclosure also provides a nucleic acid molecule encoding anoxidoreductase variant as described above.

The disclosure further provides, a vector comprising a DNA sequenceencoding the above oxidoreductase variant. The DNA sequence may beoperably linked to an expression control sequence.

In addition, the disclosure provides a host cell which is transformedwith an expression vector as described above.

A variant of an oxidoreductase apoenzyme which is substantiallyenzymatically inactive and capable of efficiently binding to a coenzymecan be obtained by introducing mutations in the amino acid sequence of acorresponding wild type enzyme. The desired properties are obtainable bysubstitutions of individual amino acids and/or by fusing additionalsegments of several amino acids at the C terminus or the N terminusand/or by inserting such segments into the amino acid sequence of adesired oxidoreductase.

Thus, the present disclosure further provides a method for obtainingsuitable variants, the method comprising

-   -   (a) providing a DNA sequence coding for a wild-type        oxidoreductase enzyme,    -   (b) introducing mutations into the DNA sequence to obtain        oxidoreductase variants,    -   (c) selecting the variants for (i) substantial lack of enzymatic        activity and (ii) capability of binding coenzyme, and        identifying and isolating suitable enzymes, and    -   (d) genetically characterizing and isolating the selected        variants.

An oxidoreductase variant of the present disclosure is suitable for usein the determination of an analyte wherein the amount of coenzyme isindicative of the amount of analyte.

The disclosure also provides a reagent kit, comprising an oxidoreductasevariant as described above and further reagents required for thedetermination of an analyte.

Further features and advantages of the invention will become apparentfrom the following discussion and the accompanying figures in which:

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the fluorescence quenching occurring with free NADHand the protection of the fluorophor NADH when surrounded by a protein;

FIG. 2 shows the DNA sequence coding for wild type glucose dehydrogenasederived from B. subtilis;

FIG. 3 shows the protein sequence of the wild type glucose dehydrogenasederived from B. subtilis;

FIG. 4 shows the screening of GlucDH mutants;

FIG. 5 shows the result of GlucDH mutant screening depicted in the formof a two dimensional landscape;

FIG. 6 shows genetic analyses of the GlucDH mutants RC-28, RC-35 andRC-21, compared to wild type GlucDH;

FIG. 7 shows a DNA sequence coding for GlucDH mutant RC-21;

FIG. 8 shows the protein sequence of GlucDH variant RC-21 (SEQ ID NO:8);

FIG. 9 shows the DNA sequence coding for GlucDH variant RC-28 (SEQ IDNO: 9);

FIG. 10 shows the protein sequence of GlucDH variant RC-28 (SEQ ID NO:10);

FIG. 11 shows the DNA sequence coding for GlucDH variant RC-35 (SEQ IDNO: 11);

FIG. 12 shows the protein sequence of GlucDH variant RC-35 (SEQ ID NO:12);

FIG. 13 shows the DNA sequence coding for BsGlucDH-His6 (SEQ ID NO: 13);

FIG. 14 shows the protein sequence of BsGlucDH-His6 (SEQ ID NO: 14) with6 His residues at the C-terminus; and

FIG. 15 shows a graph of fluorescence intensity and GlucDH enzymaticactivity of BsGlucDH, RC-21-His, RC-28-His, RC-35-His and GlucDH-His.

DETAILED DESCRIPTION

In terms of the present disclosure, “enzymatically inactive” means thatthe catalytic enzymatic activity of an oxidoreductase of the presentdisclosure is substantially eliminated. For example, the residualenzymatic activity may be about 3% or less, such as, about 2% or about1% the catalytic activity of the wild type enzyme. Further, “capable ofefficient binding to a coenzyme” means that the variant retains about10% or more of the coenzyme binding activity of the wild type enzyme asdetermined by fluorescence measurements. For example, the variant mayretain about 20% or more of coenzyme binding activity of the wild typeenzyme as determined by fluorescence measurements

Within the meaning of the present invention, coenzymes include organicmolecules which can be bound covalently or non-covalently to an enzyme.Examples of coenzymes are flavine derivatives, nicotine derivatives andquinone derivatives, for example flavine nucleoside derivatives such asFAD, FADH₂, FMN, FMNH₂, and the like, nicotine nucleoside derivativessuch as NAD⁺ (=NAD), NADH/H⁺ (=NADH), NADP⁺ (=NADP), NADPH/H⁺ (=NADPH),and the like, or ubiquinones such as coenzyme Q, PQQ, and the like.

The oxidoreductase variant of the invention can be derived from anyoxidoreductase. For example, D-lactate hydrogenase, glucose 6-phosphatedehydrogenase and glucose dehydrogenase may be utilized.

As mentioned above the variant of the oxidoreductase may be derived fromglucose dehydrogenase. Glucose dehydrogenase (GlucDH) catalyzes theoxidation of D-glucose to D-glucono-δ-lactone in the presence ofcoenzyme NAD⁺ or NADP⁺. NAD(P)⁺ dependent GlucDH is produced by Bacillusspecies during endospore formation, and has been suggested to play arole in spore germination. NAD(P)⁺ indicates that both coenzymes NAD⁺ orNADP⁺ may be used. The purified enzymes from B. cereus (Bach, J. A. andSadoff, H. L. (1962) Aerobic sporulation bacteria. I. Glucosedehydrogenase of Bacillus cereus. J. Bacteriol. 83, 699–707), B.megaterium M1286 (Pauly, H. E. and Pfleiderer, G. (1975) D-Glucosedehydrogenase from Bacillus megaterium M 1286: purification, propertiesand structure. Hoppe-Seyler's Z. Physiol. Chem. 356, 1613–1623), B.megaterium IAM1030 (Mitamura, T., Evora, R. V., Nakai, T., Makino, Y.,Negoro, S., Urabe I., and Okada, H. (1990) Structure of isozyme genes ofglucose dehydrogenase from Bacillus megaterium IAM1030. J. Ferment.Bioeng. 70, 363–369, Nagao, T., Mitamura, T., Wang, X. H., Negoro, S.,Yomo, T., Urabe, I., and Okada, H. (1992) Cloning, nucleotide sequencesand enzymatic properties of glucose dehydrogenase isozymes from Bacillusmegaterium IAM1030. J. Bacteriol. 174, 5013–5020), and B. subtilis(Makino, Y., Ding, J.-Y., Negoro, S., Urabe, I., and Okada, H. (1989)Purification and characterization of new glucose dehydrogenase fromvegetative cells of Bacillus megaterium. J. Ferment. Bioeng. 67,372–379;, Lampel, K. A., Uratani, B., Chuadhry, G. R., Ramaley, R. F.,and Rudikoff, S. (1986) Characterization of the developmentallyregulated Bacillus subtilis glucose dehydrogenase gene. J. Bacteriol.166, 238–243) have been characterized, and the GlucDH genes from B.subtilis and from different strains of B. megaterium, M1286 (Ramaley, R.F. and Vasantha, N. (1983) Glycerol protection and purification ofBacillus subtilis glucose dehydrogenase. J. Biol. Chem. 258,12558–12565., Heilmann, H. J., Mägert, H. J., and Gassen, H. G. (1988)Identification and isolation of glucose dehydrogenase genes of Bacillusmegaterium M 1286 and their expression in Escherichia coli. Eur. J.Biochem. 174, 485–490.), IWG3 (Makino, Y., Negoro, S., Urabe, I., andOkada, H. (1989) Stability-increasing mutants of glucose dehydrogenasefrom Bacillus megaterium IWG3. J. Biol. Chem. 264, 6381–6385,

Mitamura, T., Urabe, I., and Okada, H. (1989) Enzymatic properties ofisozymes and variants of glucose dehydrogenase from Bacillus megaterium.Eur. J. Biochem. 186, 389–393), and IAM1030 (Nagao, T., Mitamura, T.,Wang, X. H., Negoro, S., Yomo, T., Urabe, I., and Okada, H. (1992)Cloning, nucleotide sequences and enzymatic properties of glucosedehydrogenase isozymes from Bacillus megaterium IAM1030. J. Bacteriol.174, 5013–5020), have been cloned and the corresponding expressedenzymes characterized.

A glucose dehydrogenase variant of the present disclosure may be derivedfrom any wild-type NAD(P)⁺ dependent glucose dehydrogenase. Suitablewild-type enzymes occur in different organisms, e.g. gram-positivebacteria such as Bacillus species, including B. cereus, B. megateriumand B. subtilis.

Once bound to a glucose dehydrogenase variant according to the presentdisclosure, the coenzyme NAD(P)H can be determined by fluorescencemeasurement. This is possible because this binding results in asuppression of fluorescence quenching otherwise caused by quenchingmolecules in too close a proximity to NAD(P)H. This suppression ofquenching results in an increase of fluorescence (FIG. 1). This effectis useful for the determination of an analyte, e.g. glucose, in an assaysystem wherein the amount of coenzyme bound to a variant according tothe present disclosure, e.g. NAD(P)H present or generated in the sampleto be tested, corresponds to the amount of the analyte to be determined.

As mentioned above, one example of a wild-type enzyme from whichsuitable variants may be derived is the glucose dehydrogenase from B.subtilis (BsGlucDH), the sequence of which is shown in FIG. 3 (SEQ IDNO: 6). The catalytic activity of an oxidoreductase such as GlucDH, canbe eliminated by introducing mutations/alterations in the amino acidsequence of the enzyme. Examples of such mutations include, deletion(s),insertion(s) and substitution(s) of individual amino acids, or segmentscontaining several amino acids. Introducing these alterations result inobtaining enzyme variants which are rendered enzymatically inactivewhile being capable of efficient binding to a coenzyme.

In one particular example the enzyme is rendered enzymatically inactiveby introducing sequence alteration(s) within a region of the enzyme, forexample, defined by amino acid residues 140–165. As previously mentionedexamples of these alterations include deletion (s), insertion(s), andsubstitution(s). In one particular example, these alterations can beintroduced within the region of the enzyme defined by amino acidresidues 140–146. In another particular example, these alterations canbe introduced in the region of the enzyme defined by amino acid residues155–163. In another specific example, the enzyme BsGlucDH is renderedenzymatically inactive while being capable of efficient binding to acoenzyme by introducing alterations in one or both of the regions of theenzyme defined by amino acid residues 140–146 and 155–163. It iscontemplated that introducing such alterations in a corresponding regionof another oxidoreductase enzyme will result in the enzyme beingrendered enzymatically inactive while being capable of efficient bindingto a coenzyme. Note that within the amino acid sequence of anoxidoreductase enzyme a single amino acid may be altered, e.g.substituted or two or more amino acids may be altered, e.g. substituted,as long as the desired properties are obtained.

In the case of BsGlucDH, any one of, or both of amino acid positions 145and 158 may be substituted. For example, the serine in position 145 canbe replaced by a different amino acid, e.g. threonine. Furthermore, thetyrosine position 158 can be replaced by a different amino acid, e.g.phenylalanine. Corresponding mutations may be carried out in otherGlucDH species such as in GlucDH isolated from Bacillus megateriumstrains M1286, IWGG3, IAM1030 and IFO12108 (Itoh N, Wakita, R. 2004,Bacillus megaterium IFO12108 glucose dehydrogenase, EP1382683-A2),respectively. Examples of suitable variants of BsGlucDH are the proteinswhich have the sequences as shown in SEQ ID NOs: 8, 10 or 12. Furthervariants of BsGlucDH or GlucDH species from other organisms may beidentified by suitable screening methods as described below, e.g. bymutagenic PCR.

Furthermore, enzyme variants having the desired properties as discussedherein are obtainable by fusing an amino acid segment containing severalamino acids capable of forming a metal chelate to an amino acid sequenceof an oxidoreductase. For example, an amino acid segment capable offorming a metal chelate may be chemically bound to either or both of theC terminus and N terminus of the protein sequence of an oxidoreductaseand, in particular, of a glucose dehydrogenase. The additional segment,however, can also be inserted into the amino acid sequence of theglucose dehydrogenase, as long as the desired properties are maintained.The additional segment is capable of forming chelate complexes withmetal ions, and thus includes a plurality of amino acids with sidechains capable of chelate formation. In a further possibility thepeptide capable of forming a metal chelate complex will comprise aso-called His-tag at its C-terminal end which allows for an easypurification by well established chromatographic methods, e.g. use ofhexa-His and Ni-NTA-chromatography. For example, the addition of aplurality, e.g. 6 histidine residues at one or both of the C terminusand N terminus of the wild-type enzyme or a variant thereof results inthe formation of a coenzyme binding enzyme without catalytic activity.The amino acid sequence of a particularly suitable variant BsGlucDH-His6is shown in SEQ ID NO: 14.

Even though the mutations described in the present disclosure referparticularly to BsGlucDH, it is to be understood that they can also beused to modify corresponding sequence positions of other oxidoreductasese.g. other GlucDH. Such amino acid substitutions may be made on thebasis of, for example, similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity and the amphipathic nature of theresidues involved.

The present disclosure also relates to a DNA molecule encoding anoxidoreductase variant such as a glucose dehydrogenase variant which is(i) substantially enzymatically inactive and (ii) capable of efficientlybinding a coenzyme.

In one example, a glucose dehydrogenase variant is involved, and the DNAmolecule comprises

-   -   (a) a nucleotide sequence as shown in any of SEQ ID NOs: 7, 9,        11 or 13;    -   (b) a nucleotide sequence which encodes the same protein as (a)        but differs therefrom because of the degeneration of the genetic        codes; or    -   (c) a nucleotide sequence corresponding to the sequence of (a)        or (b) which is derived from a different species.

The identification of further oxidoreductase (e.g. GlucDH) codingsequences from suitable sources, e.g. microorganisms as well as thepurification, characterization, and cloning of relevant DNA sequencesmay be carried out based on the use of either primer or probe sequences,or a combination of these, derived from known oxidoreductase (GlucDH)sequences, for example from GlucDH sequences derived from Bacillusspecies. These primers and probes are selected from conserved sequenceportions in the GlucDH sequence and are capable of hybridizing understringent conditions to the further GlucDH sequences. Examples ofstringent conditions include detecting a positive hybridization signalafter washing for 1 h with 1×SSC and 0.1% SDS at temperatures between50° C. and 65° C., for example 55° C., 62° C. or 65° C. Particularexamples of stringent conditions include washing for 1 h with 0.2×SSCand 0.1% SDS at temperatures such as 50° C., 55° C., 62° C. or 65° C.

Further, the present disclosure relates to a vector comprising a nucleicacid sequence coding for an oxidoreductase variant such as a glucosedehydrogenase variant. In one aspect of the disclosure, the vectorincludes a DNA sequence derived from B. subtilis wild type GlucDH. Thevector may be a prokaryotic or eukaryotic vector. The vector may be anepisomal vector or a vector capable of integration into the host cellgenome. Examples of vectors are plasmids, cosmids, bacteriophage orviral vectors and artificial chromosomes.

In one instance, the vector is an expression vector wherein the codingsequence is operably linked to a promoter sequence which is capable ofdirecting its expression in a host cell. The expression vector of theinvention may further comprise an origin of replication and/or atranscription termination sequence.

Expression vectors may contain further genetic elements, e.g. an originof replication, a promoter located in front (i.e. upstream of) the DNAsequence and followed by the DNA sequence coding for all or part ofoxidoreductase variants such as, in particular, GlucDH variants. Thecoding DNA sequence may be followed by transcription terminationsequences and the remaining vector sequences. It is also possible thatthe expression vectors may also include either DNA sequences known inthe art. For example, they may further include stability leadersequences which provide for the stability of the expression product;secretory leader sequences which provide for secretion of the expressionproduct, sequences which allow expression of the structural gene to bemodulated (e.g., by the presence or absence of nutrients or otherinducers in the growth medium), marking sequences which are capable ofproviding phenotypic selection in transformed host cells, and sequenceswhich provide sites for cleavage by restriction endonucleases.

It is understood that the characteristics of the actual expressionvector used must be compatible with the host cell which is to beemployed. For example, when cloning in an E. coli cell system, theexpression vector should contain promoters which are operative in E.coli cells (e.g., tac, lac, or trp).

Suitable origins of replication for use in E. coli hosts include, forexample, a ColE1 plasmid replication origin. Suitable promoters include,for example, the tac, lac, and trp. It is also preferred that theexpression vector includes a sequence coding for a selectable marker.The selectable marker is preferably antibiotic resistance. As selectablemarkers, ampicillin resistance, or kanamycin resistance may beconveniently employed.

Suitable expression vectors containing the desired coding and controlsequences may be constructed using standard recombinant DNA techniquesas described in Sambrook et al. (“Molecular Cloning: A LaboratoryManual” (1989), Cold Spring Harbour, N.Y., Cold Spring HarbourLaboratory Press) and Ausubel, F., et al. in (“Current Protocols inMolecular Biology” (1994) John Wiley and Sons, Inc.).

This disclosure also relates to a host cell comprising an expressionvector disclosed herein, operably linked to a promoter sequence. Thehost cells are preferably transformed with an expression vector whichcomprises a DNA sequence encoding an oxidoreductase variant such as aGlucDH variant, for example. Preferred host cells include, for example,E. coli. e.g. the strains E. coli HB101 (ATCC 33694) available fromPromega (2800 Woods Hollow Road, Madison, Wis., USA) and XL1-Blueavailable from Stratagene (11011 North Torrey Pine Road, La Jolla,Calif., USA), and other prokaryotic host cells.

Expression vectors may be introduced into host cells by various methods.For example, transformation of host cells with expression vectors can becarried out by polyethylene glycol mediated protoplast transformationmethod (Sambrook, et al. 1989). However, other methods for introducingexpression vectors into host cells, for example, electroporation,ballistic injection, or protoplast fusion, can also be employed.

Further, the present invention relates to a method of obtaining improvedoxidoreductase variants and preferably improved glucose dehydrogenasevariants. For optimizing protein functions, several approaches may beused. One approach does not require specific knowledge about the enzymeitself, only about the parameters to be optimized. One screeningapproach is to use both mutation and crossover procedures synchronouslyto screen populations of variant molecules in parallel for specificoptimized parameters.

More particularly, new oxidoreductase variants which are substantiallyenzymatically inactive and capable of efficiently binding a coenzyme areobtained by a method comprising:

-   -   (a) providing a DNA sequence coding for a wild-type        oxidoreductase (glucose dehydrogenase) enzyme,    -   (b) introducing mutations to obtain oxidoreductase (glucose        dehydrogenase) variants, e.g. introducing random point mutations        into the sequence or a predetermined portion thereof, preferably        by mutagenic PCR;    -   (c) selecting the variants for        -   (i) substantial lack of enzymatic activity and        -   (ii) capability of binding coenzyme, and identifying and            isolating suitable enzymes, and (d)

The predetermined portion in step (b) may have a length of 15–50nucleotides, wherein single or multiple random point mutations may beintroduced.

Step (c) may include a determination of catalytic activity and coenzymebinding properties in a single assay, for example in afluorescence-based assay.

The oxidoreductase variants of the invention can be used for thedetermination of an analyte, wherein the amount of coenzyme, e.g.NAD(P)H, is indicative of the amount of analyte.

According to one embodiment, the oxidoreductase variants are useful in amethod for detecting an analyte in a sample, comprising

-   -   (a) contacting the sample suspected to contain the analyte with        a reagent comprising a variant of an oxidoreductase apoenzyme as        described above, under conditions wherein the amount of coenzyme        bound to the variant is indicative of the amount of analyte to        be determined,    -   (b) detecting the coenzyme-oxidoreductase variant complex.

The embodiments of the present invention make it possible toqualitatively or quantitatively determine analytes in a simple manner.Methods described herein are suitable for detecting any analyte whichcan be detected by means of an enzymatic redox reaction involving acoenzyme.

The detection reagent contains an oxidoreductase variant of thedisclosure in a quantity which is sufficient to enable the detection ofthe analyte, in accordance with the desired test format. The analyte maybe determined qualitatively and/or quantitatively. Since in one methodaccording to the disclosure, the coenzyme is detected directly, it isnot necessary for mediators or other substances which can bring aboutregeneration of the coenzyme to be present. These methods and thedetection system make it possible to use very small quantities ofsample, for example, sample volumes of <1 ml, or even <0.1 ml. Whereappropriate, the sample can also be diluted before being brought intocontact with the detection reagent. Further, very low concentrations ofa coenzyme can be detected by the method of the invention. For example,it is possible to determine analytes present in a concentration <10⁻⁴ Mor <10⁻⁵ M. These methods can be carried out without using mediatorsand/or dyes.

Examples of oxidoreductase variants for use in the detection methods areglucose dehydrogenase variants and, for example, those having thesequence as shown in any of SEQ ID NOs 8, 10, 12 or 14.

The analyte which is detected by the methods disclosed may be thecoenzyme itself, e.g. NAD(P)H/H⁺. The coenzyme, e.g. NAD(P) H/H⁺, mayalso be generated by a precedent enzymatic reaction of the analyte witha precursor of the coenzyme, e.g. NAD(P)⁺.

As the skilled person will appreciate, an inactive variant of anoxidoreductase may be used in any enzymatic assay in which the samecoenzyme is used by the enzyme on which this enzymatic procedure isbased.

Thus, any biological or chemical substance which can be determined bymeans of enzymatic reaction can be chosen as analyte, for example, anenzyme or an enzyme substrate. Examples of suitable analytes areglucose, lactic acid, malic acid, glycerol, alcohol, cholesterol,triglyceride, ascorbic acid, cysteine, glutathione, peptides, etc.

One application of the improved oxidoreductase variants described hereinis the use in test strips to monitor analyte concentrations inbiological samples, e.g. in samples from patients via determination ofNAD(P)H without quenching. Body fluids like serum, plasma, intestinalfluid or urine are examples of sources for such samples.

The oxidoreductase variants can also be used as biosensors formonitoring NAD(P)H concentrations in a reaction vessel or a reactor.

In principle, the coenzyme bound in the variant oxidoreductaseapoenzyme/coenzyme complex can be detected in any desired manner. Forexample, optical methods may be used for the detection. Opticaldetection methods include, for example, the measurement of absorption,fluorescence, circular dicroism (CD), optical rotary dispersion (ORD)and refractometry. In particular, fluorescence measurement is highlysensitive and makes it possible to detect even low concentrations of theanalyte in miniaturized systems.

The method or detection system according to the invention can comprise aliquid assay with the reagent being present, for example in the form ofa solution or suspension in an aqueous or non-aqueous liquid or as apowder or lyophilisate. However, the method and detection system mayinclude a dry assay with the reagent being applied to a support. Thesupport can include, for example, a test strip which includes anabsorbent and/or swellable material which is wetted by the sample liquidto be investigated. Examples thereof are the matrices and detectionmethods described in PCT/EP03/05177.

Further, the disclosure relates to a reagent for the determination of ananalyte in a sample, comprising an oxidoreductase variant such as aglucose dehydrogenase variant as indicated above.

Furthermore, the present disclosure relates to a reagent kit for thedetermination of an analyte in a sample comprising an oxidoreductasevariant such as a glucose dehydrogenase variant as indicated above andfurther reagents required for the determination of the analyte.

The reagent and reagent kit may be components of a liquid or dry assay.For example, they may be components of a test strip.

The above description will now be explained by way of the followingexample.

EXAMPLES

All reagents, restriction enzymes, and other materials were obtainedfrom Roche Diagnostics, unless specified from other commercial sources,and used according to the indication by suppliers. Operations employedfor the purification, characterization and cloning of DNA can be adaptedfrom published literatures.

1. Cloning and Expression of Bacillus subtilis Glucose Dehydrogenase(BsGlucDH) Gene in E. coli

The BsGlucDH gene was amplified from B. subtilis genome with PCR. Boththe amplified DNA fragment and the pKK-T5 plasmid were digested with therestriction enzymes EcoRI and HindIII. The digested products were gelpurified and ligated. An aliquot of the ligation reaction mixture wasused to transform competent E. coli cells, for example E. coli XL1 Bluecells. The transformants were subsequently selected on LB platescontaining ampicillin. To assay, individual colonies were chosen, grownover night in LB medium (cf. Sambrook et al., 1989, supra) containingampicillin and subjected to screening.

2. Mutating the Wild-Type Bacillus subtilis Glucose Dehydrogenase

In order to obtain mutant enzymes with desired properties, mutagenic PCRwas used to generate BsGlucDH. Mutagenic PCR is a method to introducerandom point mutations into the selected DNA. This method was performedaccording to the protocol described by Cadwell and Joyce (Cadwell, R. C.Joyce, G. F., PCR Methods Appl 3 (1994) 136–40) with some modifications.These modifications included different concentrations of primers, higherconcentration of MgCl₂ and different concentrations of MnCl₂ asspecified below. The Mutagenic PCR was set up as following:

Template DNA 60 fmol Forward Primer 40 pmol Reverse Primer 40 pmol 10×Taq Puffer 10 μl (without Mg²⁺; Roche #1 699 105) MgCl₂ 7 mM MnCl₂ 0–0.8mM dATP, dGTP 0, 2 mM (Roche #1 969 064) dCTP, dTTP 1 mM (Roche #1 969064) Taq DNA polymerase 5 U (Roche #1 418 432) H₂O added to total volumeof 100 μl

The PCR cycling was done with the following conditions:

5 min 95° C.,

1 min 95° C.,

1 min 50° C.,

1 min 72° C.

(30 cycles).

5 min 72° C.

The product was kept at 4° C. before analysis of the product or cloningsteps using this product were performed.

Forward primer was EF1 (5′ TTC ACA CAG GAA ACA GAA TTC ATG 3′) (SQ IDNo. 1) and reverse primer was HR1 (5′ TCC GCC AAA ACA GCC AAG CTT TTA3′) (SEQ ID NO: 2). Each round of mutagenic PCR was performed with MnCl₂concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mM MnCl₂to obtain different mutagenic rates and therefore different mutatedclones.

Mutagenic PCR products were purified using ion exchange techniques(Roche High Pure® PCR Product Purification Kit # 28104) and were elutedin H₂O. The purified PCR products and plasmid pKK-T5 were digested withrestriction enzymes EcoRI and HindIII, purified by preparative gelelectrophoresis (1% agarose/TAE) and gel extraction with QIAquick GelExtraction Kit (Quiagen Cat. # 28706). EcoRI/HindIII-digested PCRproducts were ligated into EcoRI/Hind III-digested plasmid vector pKK-T5using T4 DNA ligase (Roche # 481 220) according to the supplier'smanual. Ligation reactions were introduced into E. coli HB101 accordingto the manual for high-efficiency transformation by electroporation(Current protocols in molecular biology, chapter 1.8.4) or intoEpicurian Coli® XL1-Blue super competent cells (Stratagene # 200236)according to the supplier's manual. Transformants were plated on LB-Agarwith 100 μg/ml ampicillin and grown at 37° C., 14 h.

3. Assay and Screening of Bacillus subtilis Glucose Dehydrogenase andits Variants

The enzymatic activity assay for BsGlucDH and its variants was performedby using glucose as substrate and a coupled enzymatic reaction.

The reaction mixture was set up by combining the following reagents:

-   -   1. 2.7 ml of 0.2 M NaCl+0.1 M Tris buffer, pH 8.5    -   2. 0.2 ml of 1.1 M glucose    -   3. 0.10 ml of 15 mM NAD⁺    -   4. 0.05 ml of enzyme sample

The assay for GlucDH was carried out at 25° C., and at the wavelength of340 nm for 5 min. ΔE was calculated from the measurement points of 1 to5 min.

${{GlucDH}\mspace{14mu}{enzymatic}\mspace{14mu}{activity}} = {\frac{3.05}{ɛ \times 1 \times 0.05}\;\Delta\; A\text{/}{\min\left\lbrack {{sample}\mspace{14mu} U\text{/}{ml}\text{-}{Solution}} \right\rbrack}}$ε340=6.3 [I×mmol⁻¹×cm⁻¹]

Screening clones of BsGlucDH and its variants was carried out accordingto the above assay procedure. Each screening step was performed in96-well microtiter plates (MTPs). Colonies were picked into these platesand grown for 24 h in 200 μl of LB-medium (cf. Sambrook et al., 1989,supra). These plates are called master plates. From each master plate,90 μl sample/well was transferred to a MTP containing 10 μl reagent Bper/well (Bacterial Protein Extraction Reagent Pierce No. 78248) forcell disruption. The MTP was incubated at 25° C. for 15 min.

From the working plate 2×10 μl sample/well were transferred to two emptyMTPs. After that, one was tested for BsGlucDH enzymatic activity in thepresence of glucose and NAD⁺. The other plate was used to measure thefluorescence intensity in the presence of NADH.

In order to screen for BsGlucDH variants showing a fluorescence signal,a first determination of the crude enzyme extract thus obtained wascarried out. Using a fluorescence intensity determination reagentcontaining NADH, the fluorescence signal of the same samples was againdetermined. Any clone showing high fluorescence signal was selected as acandidate mutant.

A two dimensional landscape can be plotted. The first dimensioncorresponds to the GlucDH enzymatic activity of each clone, and thesecond dimension corresponds to the corresponding fluorescence signal ofeach clone. Any clone which has no GlucDH enzymatic activity but astrong fluorescence signal is a candidate clone. (FIG. 4–5)

4. Mutants of Bacillus subtilis Glucose Dehydrogenase with DesiredProperties

The above mentioned 2 D-screening efforts led to several BsGlucDHmutants with strong fluorescence signal in the presence of NADH butwithout glucose dehydrogenase enzymatic activity at all. The sequencealterations in these variants are shown in FIG. 6.

5. Genetic Characterization of BsGlucDH Mutants with Desired Properties

The plasmid containing the mutant BsGlucDH gene and its variants wereisolated (High Pure Plasmid Isolation Kit, Roche 1754785) and sequencedusing an ABI Prism Dye Terminator Sequencing Kit and ABI 3/73 and 3/77sequencer (Amersham Pharmacia Biotech). The following primers were used:

T5 forward primer 5′-GCC ATC TCA CGA AAT GCC-3′ (SEQ ID NO:3) T5 reverseprimer 5′-ATT GTT CAC GCG AAT GCC-3′ (SEQ ID NO:4)

Preferred BsGlucDH mutants with improved properties were geneticallycharacterized. The DNA and corresponding amino acid sequences are givenin FIG. 7–12. Amino acid substitutions of the BsGlucDH variants withdesired properties are mutations: Y158F in mutant RC21 (SEQ ID NOs 7/8),S145T in mutants RC28 (SEQ ID NOs 9/10) and RC35 (SEQ ID NOs 11/12). Thewild-type BsGlucDH and RC21, RC28, and RC35 with 6 His residues at theC-terminus were also generated. These enzymes were designatedGlucDH-His6, RC21-His6, RC28-His6 and RC35-His6. The DNA andcorresponding amino acid sequence of the His6-wild type enzyme (SEQ IDNOs 13/14) are given in FIGS. 13 and 14.

6. Purification of the BsGlucDH Variants RC21, RC28 and RC35

The isolation and purification of BsGlucDH and BsGlucDH variants asselected above from cell cultures can be carried out by any knownmethod, such as the following. After the cells were cultured in anutrient medium, they were recovered by centrifugation. The cell pelletswere homogenized in 20 mM Tris-HCl (pH 8) using high pressure celldisruption at a pressure of 900 bars to give a crude cell extract. Thecell extract containing the enzyme was precipitated with Polymin G20(0.4%), and subsequently precipitated with 2.0M (NH₄)₂SO₄. The extractwas then subjected to phenyl sepharose column chromatography (PharmaciaBiotech) to give a standard purified enzyme product. Usually, the endproduct thus obtained was at least 90% pure and shows one predominantband in SDS-PAGE corresponding to BsGlucDH. In case SDS PAGE shouldoccasionally reveal a lower degree of purity the purification steps maybe repeated.

7. Characterization of BsGlucDH and its Variants

The characterizations of BsGlucDH and its variants included a)determination of the enzymatic activity in the presence of NAD, and b)determination of the fluorescence signal in the presence of NADH.

a) Determination of the Enzymatic Activity in the Presence of NAD

For determination of the enzymatic activity, the purified enzyme orenzyme mutant was assayed according to the assay procedure described inExample 3

b) Determination of the Fluorescence Signal in the Presence of NADH

The determination of the fluorescence signal in the presence of NADH wasdone with purified enzyme or enzyme mutants. The enzyme or enzyme mutantwas used at a concentration of 1.0×10⁻⁵ M. NADH was added at aconcentration of 1.0×10⁻⁵ M. The incubations were carried out in 0.2 MNaCl+0.1 M Tris buffer at pH 8.5. The fluorescence measurement was setup as the following: Excitation at 360 nm, Emission at 465 nm, 10reading cycles with an interval of 5 seconds between each cycle.

As shown in FIG. 15, all mutants RC21-His6, RC28-His6, RC35-His6 andGlucDH-His6 have no glucose dehydrogenase enzymatic activity at all, butat the same time can still bind to NADH and give a strong fluorescencesignal.

All references referred to in this disclosure are expressly incorporatedherein.

1. A glucose dehydrogenase variant having about 3% or less residualenzymatic activity relative to the wild type enzyme and which retainsabout 20% or more of the coenzyme binding activity of the wild typeenzyme, wherein said variant comprises an amino acid substitution atposition 145 or 158 of the amino acid sequence of SEQ ID NO: 6 of B.subtilis.
 2. The glucose dehydrogenase variant as claimed in claim 1,wherein the coenzyme is NAD(P)H.
 3. The glucose dehydrogenase variant asclaimed in claim 1 which has a threonine substitution at position 145 ora phenylalanine substitution at position 158 of the amino acid sequenceof SEQ ID NO: 6 of B. subtilis.
 4. The glucose dehydrogenase variant asclaimed in claim 3, having fused thereto an amino acid segment capableof forming a metal chelate.
 5. The glucose dehydrogenase variant asclaimed in claim 4, wherein the amino acid sequence of said variantcomprises the sequence of SEQ ID NO:
 14. 6. A glucose dehydrogenasevariant comprising the amino acid sequence as shown in SEQ ID NO:
 8. 7.The glucose dehydrogenase of claim 6 further comprising an amino acidsequence fused thereto that is capable of forming a metal chelate.
 8. Aglucose dehydrogenase variant comprising the amino acid sequence asshown in SEQ ID NO:
 10. 9. The glucose dehydrogenase of claim 8 furthercomprising an amino acid sequence fused thereto that is capable offorming a metal chelate.